CONICET | Buscador de Institutos y Recursos Humanos

Enterocin CRL35 and Pediocin PA-1 are pediocin-like bacteriocins that act on Gram-positive bacteria membrane. In order to study the mechanism by which they induce loss of membrane integrity, in previous work we constructed "suicide probes": fused genes of bacteriocins with EtpM, a bitopic membrane protein. We demonstrated that these bacteriocins are able to exert a bactericidal effect on Gram-negative bacteria when anchored in the membrane, regardless of their specific receptor (Man-PTS system).The aim of this study was to evaluate the effect of these bacteriocins on cell membrane potential and permeability, when expressed as suicide probes in Gram-negative bacteria such as E. coli.On the one hand, we employed the potentiometric indicator DiSC3(5), a fluorophore that exhibits changes in fluorescence intensity. These changes are dependent on transmembrane potential 1, and they are feasibly to be measured over time. The results showed that the expression of fusions EtpM-bacteriocins generates potential dissipation by membrane depolarization. In contrast, neither the control strain that expressed only EtpM (membrane anchor), nor the strain that co-expresses the suicide probe with Enterocin CRL35 immunity protein (MunC) showed a significant change in membrane potencial.On the other hand, we used LIVE/DEAD BacLight kit, which includes two fluorophores that penetrate the membrane according to its integrity. This allows to discriminate between live and dead cells2. The images obtained by fluorescence microscopy clearly evinced that suicide probes expression disturbs membrane permeability, so bacteria emitted red fluorescence (dead cells), while control and co-expressing EtpM-bacteriocin/MunC strains conserve membrane integrity, so they emitted green fluorescence (live cells).